Methods of inhibiting photoreceptor apoptosis

ABSTRACT

The present invention provides methods to prevent photoreceptor death. In particular, the present invention provides peptides which prevent FAS-mediated photoreceptor apoptosis.

The present application is a divisional of pending U.S. patentapplication Ser. No. 12/716,986 filed Mar. 3, 2010, which claimspriority to U.S. Provisional Application Ser. No. 61/157,079, filed Mar.3, 2009, and U.S. Provisional Application Ser. No. 61/254,082, filedOct. 22, 2009, which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention provides methods to prevent photoreceptor death.In particular, the present invention provides peptides which preventFAS-mediated photoreceptor apoptosis.

BACKGROUND OF THE INVENTION

Apoptosis (programmed cell death) plays a central role in thedevelopment and homeostasis of all multi-cellular organisms. Alterationsin apoptotic pathways have been implicated in many types of humanpathologies, including developmental disorders, cancer, autoimmunediseases, as well as neuro-degenerative disorders, and retinaldegradation. It is a tightly regulated pathway governing the deathprocesses of individual cells and can be initiated either extrinsicallyor intrinsically. The latter is an intracellular mechanism triggered bythe mitochondria while the former involves the interaction of a ‘deathreceptor’ with its corresponding ligand at the cell membrane.

Thus, the programmed cell death pathways have become attractive targetsfor development of therapeutic agents. In particular, since it isconceptually easier to kill cells than to sustain cells, attention hasbeen focused on anti-cancer therapies using pro-apoptotic agents such asconventional radiation and chemotherapy. These treatments are generallybelieved to trigger activation of the mitochondria-mediated apoptoticpathways. However, these therapies lack molecular specificity, and morespecific molecular targets are needed.

Retinal detachment (RD), defined as the separation of the neurosensoryretina from subjacent retinal pigment epithelium (RPE), results in theapoptotic death of photoreceptor cells (Cook et al. 1995; 36(6):990-996;Hisatomi et al. Curr Eye Res. 2002; 24(3):161-172; Zacks et al. InvestOphthalmol Vis Sci. 2003; 44(3):1262-1267. Yang et al. Invest OphthalmolVis Sci. 2004; 45(2):648-654; herein incorporated by reference in theirentireties). Rodent and feline models of RD have demonstrated theactivation of pro-apoptotic pathways nearly immediately after the retinabecomes separated from the RPE (Cook et al. 1995; 36(6):990-996;Hisatomi et al. Curr Eye Res. 2002; 24(3):161-172; Zacks et al. InvestOphthalmol Vis Sci. 2003; 44(3):1262-1267. Yang et al. Invest OphthalmolVis Sci. 2004; 45(2):648-654; herein incorporated by reference in theirentireties). Histological markers of apoptosis such as terminaldeoxynucliotidyl transferase nick end label (TUNEL) staining reach apeak at approximately three days after RD, with apoptotic activity andprogressive cell death persisting for the duration of the detachmentperiod. Clinical experience in the repair of retinal detachments,however, has demonstrated that there is a window of opportunity forrepair with preservation of good visual acuity. Retrospective caseseries have demonstrated that significant numbers of patients withmacula-off RDs repaired within 5-10 days after onset of detachment canretain relatively good visual function, but that the visual acuity dropssignificantly as the time between detachment and repair extends (Burton.Trans Am Ophthalmol Soc. 1982; 80:475-497; Ross et al. Ophthalmology.1998; 105(11):2149-2153; Hassan et al. Ophthalmology. 2002;109(1):146-152; herein incorporated by reference in their entireties).The delayed time between the activation of pro-apoptosis pathways andthe clinical onset of visual loss suggests that intrinsicneuroprotective factors may become activated within the neural retina,and may serve to counter-balance the effects of the pro-apoptoticpathways activated by retinal-RPE separation.

SUMMARY

In some embodiments, the present invention provides a method ofinhibiting photoreceptor apoptosis comprising administering aphotoreceptor protective composition. In some embodiments, thephotoreceptor protective composition comprises photoreceptor protectivepolypeptide or a nucleic acid encoding a photoreceptor protectivepolypeptide. In some embodiments, the photoreceptor protectivepolypeptide comprises IL-6 or a fragment thereof. In some embodiments,the photoreceptor protective polypeptide comprises XIAP or a fragmentthereof. In some embodiments, the photoreceptor protective polypeptidecomprises MET or a fragment thereof. In some embodiments, the fragmentof MET comprises MET12. In some embodiments, the fragment of METcomprises at least 70% (e.g., at least 80%, 85%, 90%, 95%) sequencesimilarity to MET12. In some embodiments, the photoreceptor apoptosiscomprises FAS-mediated photoreceptor apoptosis. In some embodiments, thephotoreceptor protective composition is administered to a population ofcells. In some embodiments, the photoreceptor protective composition isadministered in an amount sufficient to attenuate cell death within thepopulation of cells. In some embodiments, the photoreceptor protectivepolypeptide is administered to a subject. In some embodiments, thesubject suffers from retinal detachment. In some embodiments, thesubject is at risk of retinal detachment. In some embodiments, thephotoreceptor protective composition is administered in an amountsufficient to attenuate cell death within the subject.

In some embodiments, the present invention provides a method ofincreasing photoreceptor survival comprising administering aphotoreceptor protective composition. In some embodiments, increasingphotoreceptor survival comprises inhibiting photoreceptor apoptosis. Insome embodiments, the photoreceptor protective composition comprisesphotoreceptor protective polypeptide or a nucleic acid encoding aphotoreceptor protective polypeptide. In some embodiments, thephotoreceptor protective polypeptide comprises IL-6 or a fragmentthereof. In some embodiments, the photoreceptor protective polypeptidecomprises XIAP or a fragment thereof. In some embodiments, thephotoreceptor protective polypeptide comprises MET or a fragmentthereof. In some embodiments, the fragment of MET comprises MET12. Insome embodiments, the fragment of MET comprises at least 70% sequencesimilarity to MET12. In some embodiments, the photoreceptor apoptosiscomprises FAS-mediated photoreceptor apoptosis. In some embodiments, thephotoreceptor protective composition is administered to a population ofcells. In some embodiments, the photoreceptor protective composition isadministered in an amount sufficient to attenuate cell death within saidpopulation of cells. In some embodiments, the photoreceptor protectivecomposition is administered in an amount sufficient to enhancephotoreceptor survival within said population of cells. In someembodiments, the photoreceptor protective composition is administered toa subject. In some embodiments, the subject suffers from an ocularcondition, disease, or condition or disease affecting ocular health. Insome embodiments, the subject is at risk of an ocular condition,disease, or condition or disease affecting ocular health. In someembodiments, the ocular condition, disease, or condition or diseaseaffecting ocular health comprises retinal detachment, maculardegeneration, retinitis pigmentosa, occular inflammation, autoimmuneretinopathy, trauma, cancer, tumor, uveitis, hereditary retinaldegeneration, diabetic retinopathy, choroidal neovascularization,retinal ischemia, pathologic myopia, angioid streaks, macular edema, orcentral serous chorioretinopathy. In some embodiments, the ocularcondition, disease, or condition or disease affecting ocular healthcomprises retinal detachment. In some embodiments, the ocular condition,disease, or condition or disease affecting ocular health comprisesmacular degeneration. In some embodiments, the photoreceptor protectivecomposition is administered in an amount sufficient to attenuate celldeath within said subject. In some embodiments, the photoreceptorprotective composition is administered in an amount sufficient toenhance photoreceptor survival within said subject.

In some embodiments, the present invention provides a compositioncomprising a photoreceptor protective composition and a pharmaceuticalcarrier configured for optical delivery. In some embodiments, thephotoreceptor protective composition comprises a photoreceptorprotective polypeptide or a nucleic acid encoding a photoreceptorprotective polypeptide. In some embodiments, the photoreceptorprotective polypeptide comprises IL-6 or a fragment thereof. In someembodiments, the photoreceptor protective polypeptide comprises XIAP ora fragment thereof. In some embodiments, the photoreceptor protectivepolypeptide comprises MET or a fragment thereof. In some embodiments,the fragment of MET comprises MET12. In some embodiments, the fragmentof MET comprises at least 70% (e.g., at least 80%, 85%, 90%, 95%)sequence similarity to MET12. In some embodiments, the photoreceptorapoptosis comprises FAS-mediated photoreceptor apoptosis. In someembodiments, the pharmaceutical carrier is configured for injection intothe eye of a subject. In some embodiments, the pharmaceutical carrier isconfigured for subretinal injection. In some embodiments, thepharmaceutical carrier is configured for topical application onto theeye of a subject.

In some embodiments, the present invention comprises a kit comprising aphotoreceptor protective composition and one or more additioncompositions. In some embodiments and additional compositions comprises,a photoreceptor protective composition, pharmaceutical carrier, drug,pain reliever, anesthetic, antiapoptotic agent, etc. In someembodiments, a kit comprises one or more photoreceptor protectivecomposition and other agents configured for co-administration to cellsor a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary and detailed description is better understood whenread in conjunction with the accompanying drawings which are included byway of example and not by way of limitation.

FIG. 1 shows Western blot analysis of levels of activated forms of STAT1and STAT3 in attached and detached retinas. Leftmost 2 lanes: One dayafter detachment. Middle 2 lanes: Three days after detachment. Rightmost2 lanes: Seven days after detachment. Retina-RPE separation was createdin the left eye. Attached retina was obtained from the contralateral eyeof the same animal. Equal loading was verified across all lanes.

FIG. 2 shows TUNEL staining in wildtype vs. IL6−/− mouse retinasharvested 3 days after detachment. A, B: Wildtype mice. C, D: IL-6−/−mice. A, C: Fluorescein isothiocyanate (FITC) fluorescence of TUNELpositive cells. B, D: Propidium iodide (PI) fluorescence of all nuclei.E: Graph summarizing TUNEL staining of wildtype and IL-6−/− mouseretinas 3 days after detachment. Results are means±standard error of themean.

FIG. 3 shows outer nuclear layer cell counts in wildtype vs. IL6−/− miceafter retinal detachment. A-C: Wildtype mice. D-F: IL-6−/− mice. A, D:Attached retinas. B, E: Retinas harvested 1 month after creation of thedetachment. C, F: Retinas harvested 2 months after creation of thedetachment. G: Graph summarizing ONL cell counts/total retinal thicknessin wildtype and IL-6−/− mice 1 and 2 months after retinal detachment.Results are means±standard error of the mean.

FIG. 4 shows TUNEL staining of detached rat retinas treated with IL-6neutralizing antibody or exogenous IL-6. A, B: Subretinal injection ofvehicle only at the time of creation of the detachment. C, D: Subretinalinjection of 0.15 mg anti-IL-6 NAB at the time of creation of thedetachment. E, F: Subretinal injection of 15 ng exogenous IL-6 at thetime of creation of the detachment. A, C, E: FITC fluorescence of TUNELpositive nuclei. B, D, F: PI fluorescence of all nuclei. G: Graphsummarizing effects of subretinal anti-IL-6 NAB and exogenous IL-6 onTUNEL staining of rat retinas 3 days after detachment. Results aremeans±standard error of the mean.

FIG. 5 shows Effects of IL-6 neutralizing antibody vs. exogenous IL-6 onrat retina outer nuclear layer cell counts. A: Attached retina. B, C:Retina harvested 1 and 2 months after subretinal injection of vehicleonly at the time of creation of the detachment, respectively. D, E:Retina harvested 1 and 2 months after subretinal injection of 0.15 μganti-IL-6 NAB at the time of creation of the detachment, respectively.F, G: Retina harvested 1 and 2 months after subretinal injection of 15ng exogenous IL-6 at the time of creation of the detachment,respectively. H: Graph summarizing effects of subretinal anti-IL-6 NABand exogenous IL-6 on outer nuclear layer cell count of rat retinas 1and 2 months after retinal detachment. Results are means±standard errorof the mean.

FIG. 6 shows Caspase 3 and 9 assays following retinal detachment in GFPand XIAP-treated retinas. Subretinal injections of rAAV-GFP or rAAV-XIAPwere followed by retinal detachment in the left eye (OS) of the animalRight eyes (OD) served as intact controls.

FIG. 7 shows Immunohistochemistry with antibodies to GFP (A) and to theHA tag of XIAP (B) confirmed robust over-expression in the cell bodiesand inner (IS) and outer (OS) segments of the photoreceptors from bothrAAV constructs. No primary antibody controls for GFP and XIAP are shownin C and D, respectively. TUNEL analysis confirmed that GFP-treatedretinas had more apoptotic nuclei than XIAP-treated retinas (brownpigment in E, F and black arrows in insets). TUNEL-positive pixel counts(boxplot, G) supported the immunohistochemistry results. Each boxcontains the values between the 25th and 75th percentile, and the linewithin the box represents the median value. Bar lines above and beloweach box indicate the 90th and 10^(th) percentiles, respectively. Theboxplot was generated with SigmaPlot, version 8.0 (SPSS, Inc.). ONL,outer nuclear layer.

FIG. 8 shows immunohistochemistry for GFP (A, B) and XIAP (D, E)confirmed sustained expression at 2 months after the detachment. The GFPsignal (green) was faint because many of the photoreceptors expressingthe viral transgene had died. In contrast, XIAP signal (red) was bright,and accompanied by increased numbers of photoreceptors. Note that inretinal areas where XIAP signal was reduced (arrowhead), photoreceptorloss was considerable. Rhodopsin staining (red) in GFP-injected (C) andXIAP-injected (F) retinas shows that the preserved photoreceptors areable to synthesize functional protein. Outer nuclear layer identified byarrow. Magnification bar=50 mm.

FIG. 9 shows Comparison between attached (A, C) and detached (B, D)retinas in XIAP and GFPtreated animals. At two months after detachment,XIAP-treated retinas (D) were consistently thicker than GFP-treatedretinas (B) and their inner and outer segments were more organized. Aratio was obtained by dividing the number of nuclear layers in the ONLin a detached region of the eye by the number of nuclear layers in theONL in the attached retina in the same eye (E). Magnification bar=50 mm.

FIG. 10 shows a graph of the effects of MET on the number of TUNELpositive cells post retinal detachment.

FIG. 11 shows a graph of the effect of MET on caspase activity inducedby retinal detachment.

FIG. 12 shows Fas-induced caspase 8 activation is blocked by Met12 in661W cells. A) 661W cells were treated with various concentrations ofFas-activating antibody (Fas-Ab). Caspase 8 activity was measured at 48hours. Compared with no treatment, the increase in caspase 8 activitywas statistically significant for all concentrations of Fas-Ab. B) 661Wcells were treated with 500 ng/ml of Fas-Ab and caspase 8 activity wasmeasured at various time points. Data was normalized to untreatedcontrols at each time point. C) 661W cells were treated with 500 ng/mlFas-Ab in the presence of Met12, mMet or vehicle (DMSO). Control groupdid not have Fas-Ab treatment. Caspase 8 activity was measured at 48hours.

FIG. 13 shows Met12 inhibits activation of caspases. A) Injection of theMet12 into the subretinal space of detached retinas reduces caspase 8activity. Rat retinas were detached in the presence of Met12 (50 μg),mMet (50 μg), or vehicle (DMSO). Caspase 8 activation was measured inharvested retinas after 24 hours of detachment as described. Data ispresented as fold increase in caspase activity in detached retinascompared to attached retinas. Western blot shows decreased caspase 8cleavage in the presence of Met12 (inset). Assay controls including nolysate samples (blank) and recombinant caspase 8 are shown. B) Caspase 3activity was significantly reduced by Met12 treatment during retinaldetachment. C) Caspase 9 activity was also significantly reduced byMet12. Data is presented as fold increase in caspase activity indetached retinas compared to attached retinas. Assay controls includingno lysate samples (blank) and recombinant caspase 8 or caspase 9 areshown.

FIG. 14 shows Met12-mediated inhibition of Fas-pathway signalingprevents photoreceptors from entering the apoptotic cascade. Rat retinaswere detached in the presence of Met12 (50 μg), mMet (50 μg), or vehicle(DMSO). Eyes were enucleated after 72 hours and retinas were sectionedfor TUNEL staining. A) Representative photomicrographs of TUNEL stainedphotoreceptors after 72 hours of retinal detachment. Nuclei of retinalcells are stained with propidium iodide (PI). INL: Inner nuclear layer,ONL: Outer nuclear layer. B) Quantification of TUNEL positive cells inthe ONL, mean±S.E., n=3-6. There was no TUNEL stained cells in the ONLof attached retinas.

FIG. 15 shows decrease in caspase activation and the numberTUNEL-positive cells correspond to increased long-term survival ofphotoreceptors. Retinas were detached in the presence of Met12 (50 μg),mMet (50 μg), or vehicle (DMSO), which were injected in the subretinalspace at the time of detachment. Eyes were enucleated after 2 months ofdetachment and paraffin sections were stained with 0.5% toluidine blue.A) Representative photomicrographs, INL: Inner nuclear layer, ONL: Outernuclear layer, B) ONL cell counts normalized to retinal thickness, C)ONL thickness normalized to retinal thickness.

DEFINITIONS

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 12 residue oligonucleotide is referred to as a “12-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., photoreceptor protective composition) sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages andis not intended to be limited to a particular formulation oradministration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g.,compositions of the present invention) to a subject (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplaryroutes of administration to the human body can be through the eyes(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g.,intravenously, subcutaneously, intratumorally, intraperitoneally, etc.)and the like.

As used herein, the terms “co-administration” and “co-administer” referto the administration of at least two agent(s) (e.g., photoreceptorprotective peptides, oligonucleotides coding for a photoreceptorprotective composition, and one or more other agents) or therapies to asubject. In some embodiments, the co-administration of two or moreagents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., photoreceptor protectivecomposition) with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vitro, in vivoor ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

DETAILED DESCRIPTION OF EMBODIMENTS

Experiments performed during development of embodiments of the presentinvention demonstrate that IL-6 is a controller of photoreceptorapoptosis after separation of the neurosensory retina from theunderlying RPE. Inhibition of IL-6 with genetic ablation or viaadministration of an IL-6 neutralizing antibody significantly increasedthe rate of photoreceptor apoptosis. One month after detachment, boththe IL-6−/− mice and the rats receiving subretinal IL-6 NAB hadsignificantly decreased ONL cell counts compared to their respectivecontrols. These data indicate that IL-6 is necessary for the survival ofphotoreceptors after detachment from the RPE.

Experiments were performed during development of embodiments of thepresent invention examining the effect of increasing IL-6 levels hadmixed effects, depending on the time point examined and the assaymeasured. When compared to injection of vehicle only, subretinalinjection of exogenous IL-6 did not significantly affect the percentageof TUNEL positive cells 3 days after RD, but did significantly increasethe number of photoreceptors that survived longer term detachments.Subretinal IL-6 levels in patients with RD are higher than in thevitreous of controls, and the amount of subretinal IL-6 in patients withRD is highest at 5-8 weeks following RD (Bakunowicz-Lazarczyk et al.Ophthalmologica. 1999; 213(1):25-29, herein incorporated by reference inits entirety). IL-6 signals through a combination of its ligand bindingsubunit (gp80, also known as IL-6R) and a common signal transducingsubunit (gp130) (Heinrich et al. Ann NY Acad Sci. 1995; 762:222-236,herein incorporated by reference in its entirety). Administration ofexogenous IL-6 alone does not have as much anti-apoptotic activity asadministration of IL-6 in conjunction with a soluble form of IL6-R(Inomata et al. Biochem Biophys Res Commun 2003; 302(2):226-232, Curnowet al. J Immunol. 2004; 173(8):5290-5297, herein incorporated byreference in their entireties).

Despite the activation of endogenous IL-6, there is still a relativelylinear rate of cell loss. The rate of this death is significantlydecreased by the addition of exogenous IL-6. There was significantlygreater preservation of photoreceptors in the group of rats treated withexogenous IL-6 at the time of detachment compared to the control groupat the 1 month after RD, the difference was lost 2 months after RD dueto accelerated photoreceptor loss in the exogenous IL-6 group in thesecond month. Reinjection of exogenous IL-6 at the one-month time pointsuggests that the duration of the effect of IL-6 can be extended, byallowing for a greater therapeutic “window of opportunity” to achieveretinal-RPE re-attachment.

Retinal detachments are often present for an unknown duration of timeprior to presenting to the ophthalmologist. Delay of initial subretinalinjection of exogenous IL-6 by 2 weeks from the creation of thedetachment still allowed significantly greater preservation ofphotoreceptors 1 month after detachment as compared to controls,indicating that IL-6 may still be useful in preserving photoreceptorsdespite delayed presentation of patients. The effect of a singleinjection of IL-6 2 weeks after retinal-RPE separation lasted for 2weeks.

IL-6 is known to be a strong activator of the Janus kinase (JAK)/signaltransducer and activator of transcription (STAT) pathway (Samardzija etal. FASEB J. 2006; 20(13):2411-2413, herein incorporated by reference inits entirety). Retinal-RPE separation potently activates STAT1 andSTAT3. STAT1 is associated with tumor suppression and pro-apoptoticactivity whereas STAT3 is predominantly associated with cellularproliferation and considered to be anti-apoptotic (Samardzija et al.FASEB J. 2006; 20(13):2411-2413, Aaronson et al. Science. 2002;296(5573):1653-1655. Stephanou et al. Int J Exp Pathol. 2003;84(6):239-244, Stephanou et al. Growth

Factors. 2005; 23(3):177-182, Battle et al. Curr Mol Med. 2002;2(4):381-392, herein incorporated by reference in their entireties). TheIL-6 effect is mediated predominantly through STAT3 and not STAT1.Modulation of apoptotic pathways by STATs may be though the downstreamtranscriptional regulation of factors that trigger cell death, such asFAS and caspases, or factors that promote cell survival, such as Bcl-xLand FLICE (FADD (Fas-associated death domain)-likeinterleukin-1β-converting enzyme) inhibitory protein (FLIP) (Haga et al.J Clin Invest. 2003; 112(7):989-998, Budd et al. Nat Rev Immunol. 2006;6(3):196-204, herein incorporated by reference in their entireties).FLIP is an enzymatically inactive homologue of caspase-8 that cancompete with caspase-8 for recruitment to death-inducing signalingcomplexes (DISCs), and thus acts as a dominant negative inhibitor ofapoptosis. 34 IL-6 may stabilize protein levels of FLIP as FLIP is morerapidly degraded in IL-6−/− mice (Kovalovich et al. J Biol Chem. 2001;276(28):26605-26613, herein incorporated by reference in its entirety).

Studies have shown that IL-6 may protect against retinal degenerationinduced by a range of insults including ischemia-reperfusion injury,NDMA toxicity, pressure induced death, and retinal detachment (Sanchezet al. Invest Ophthalmol Vis Sci. 2003; 44(9):4006-4011, Inomata et al.Biochem Biophys Res Commun 2003; 302(2):226-232, Sappington et al.Invest Ophthalmol Vis Sci. 2006; 47(7):2932-2942, herein incorporated byreference in their entireties). The photoreceptor-protective role ofIL-6 in the context of retinal-RPE separation suggests that this is avaluable point of therapeutic intervention for improving visual outcomein patients with this type of retinal injury.

Rapid re-attachment is imperative in order to achieve a good visualoutcome following retinal detachment. Animal studies, indicate thatcaspases—the executors of apoptosis—are activated within 24 hours aftera detachment. Patients generally do not recover visual acuity of 20/20if the duration of retinal detachment lasted 5 days or longer (Burton.Trans Am Ophthalmol Soc 1982; 80:475-497, herein incorporated byreference in its entirety). In many disease processes the reappositionof the retina to the RPE cannot be achieved quickly, resulting in thecontinuous apoptotic death of photoreceptors. The use of photoreceptorprotective agents can potentially limit the extent of photoreceptordeath until re-attachment can occur.

Experiments performed during development of embodiments of the presentinvention demonstrate that X-linked inhibitor of apoptosis (XIAP) canprotect photoreceptors for at least 2 months of continual detachment.XIAP-treated retinas maintained larger numbers of nuclear layers in theONL, and their inner and outer segments were better organized. Inaddition, they stained robustly with an antibody to rhodopsin,suggesting that they remained viable.

The protective effects of XIAP suggest blocking caspases is effective inblocking the cell death pathway. However, we cannot rule out thepossibility that XIAP is having other effects in addition to caspaseinhibition, although an understanding of the specific mechanism ofaction is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action.XIAP has been shown to suppress cell death via other mechanisms. Throughits RING zinc finger domain, XIAP has E3 ubiquitin ligase activity andcan promote the degradation of proapoptotic proteins (reviewed in 10).XIAP is also involved in the transcriptional activation of prosurvivalpathways through TAK1 (Hofer-Warbinek et al. J Biol Chem 2000;275:22064-22068, Sanna et al. Mol Cell Biol 2002; 22:1754-1766, hereinincorporated by reference in their entireties). TAK1 is amitogen-activated protein kinase kinase kinase (MAPKKK) involved in theactivation of both the NF-kB and JNK1 prosurvival pathways. Thus, theability of XIAP to protect photoreceptors for up to 2 months may beattributed, in part, to the activation or suppression of multiplepathways. The inhibition of caspase activity and the decreased TUNELcounts in XIAP-transfected eyes, however, does support caspaseinhibition as a principal mechanism through which XIAP exerts itsphotoreceptor protective effects.

Experiments performed during development of embodiments of the presentinvention demonstrate XIAP efficacy in the treatment of retinaldetachment. Rapid delivery of XIAP to the site of retinal detachment hasthe potential to limit the acute damage suffered by photoreceptors, thusbuying the patient critical time until successful re-attachment can beachieved

Experiments conducted during development of embodiments of the presentinvention provided a method of inhibiting Fas signaling and preventingphotoreceptor apoptosis after retinal detachment. For example,experimental results demonstrated that a small peptide inhibitor of theFas death receptor blocks caspase activation and increases photoreceptorsurvival after retina-RPE separation. For example, in an in vitro modelof cone photoreceptors, the small peptide Met12 prevents theFas-dependent activation of caspase 8. Met12 significantly reducedFas-signaling and photoreceptor apoptosis in an vivo model ofexperimental retinal detachment.

Retinal detachment activates the Fas-receptor (Zacks et al. ArchOphthalmol 2007; 125:1389-1395, Zacks et al. IOVS 2004;45(12):4563-4569.8, herein incorporated by reference in theirentireties), and that this event controls activation of the intrinsiccell death pathway in photoreceptors. Photoreceptor apoptosis can beprevented using large molecules, such as neutralizing antibodies, toinhibit the Fas-receptor, or by preventing the detachment-inducedincrease in the Fas-receptor transcript with inhibitory RNA. Experimentsconducted during development of embodiments of the present inventiondemonstrated that the same significant level of photoreceptorpreservation using a small peptide, Met12.

A rodent model was used to investigate molecular mechanisms regulatingphotoreceptor death after retinal detachment. Further, Fas-Ab treatmentactivates caspase 8 in 661W cells in vitro in a dose and time-dependentmanner, demonstrating that Fas receptor signaling pathway is intact inthese cells. The 661W cell line is a functional in vitro model ofphotoreceptor apoptosis mediated by Fas signaling.

Methods herein demonstrate significant levels of photoreceptor survivalafter Met12-mediated Fas inhibition. Thus, Fas inhibition by Met12resulted in significant preservation of photoreceptor cells in vivo. Thedose of Met12 that was injected in the subretinal space at the time ofretinal detachment may be optimized for complete protection usingmethods disclosed herein. In 661W cells, where Met12 reached all cells,Met12 treatment resulted in full inhibition of the caspase 8 activation.Subretinal Met12 may not adequately reach detached photoreceptors thatare apoptotic. It is contemplated that improved administration of thepeptide yields improved photoreceptor survival. Met12 was injectedsubretinally only during the creation of retina/RPE separation. Theamount of Met12 available in the subretinal space most likely decreaseswith time, thus increasing the number of Fas receptors available forFasL binding. This is similar to what occurs with anotherneuroprotectant, IL-6. Exogenous IL-6 increases the survival of detachedphotoreceptors (Chong et al. Invest Ophthalmol Vis Sci. 2008;49(7):3193-3200, herein incorporated by reference in its entirety). WhenIL-6 was injected in the subretinal space at the time of detachment, itsprotective effect on detached photoreceptors lasted for one month.However, re-injection of exogenous IL-6 at the one-month time pointextended duration of photoreceptor survival after retina/RPE separation.It is contemplated that an optimal treatment strategy and increasedpro-survival effect of Met12 allows for a greater therapeutic “window ofopportunity” to achieve the highest photoreceptor survival rate afterretinal-RPE re-attachment.

It is contemplated that a population of photoreceptors executesapoptosis independent of Fas death receptor signaling. Theseparation-induced activation of the mitochondrial apoptotic pathway isonly partially controlled by the activation of Fas (Zacks et al. ArchOphthalmol 2007; 125:1389-1395, herein incorporated by reference in itsentirety). It is contemplated that a second and alternate signalingpathway may play a role in stimulating the intrinsic death pathway inphotoreceptors. Intrinsic pathway caspases play a critical role inretinal detachment-induced photoreceptor apoptosis (Zadro-Lamoureux etal. Invest Ophthalmol Vis Sci. 2009; 50(3):1448-1453, hereinincorporated by reference in its entirety). Delivery of X-linkedinhibitor of apoptosis (XIAP) with a recombinant adenoassociated virusinhibited caspase 3 and caspase 9 activities and protectedphotoreceptors from detachment-induced apoptosis. In addition to themitochondrial apoptotic pathway, caspase-independent death pathways mayplay a role in photoreceptor loss after retinal detachment. Activationof the apoptosis-inducing factor (AIF)-dependent death in anexperimental rat model of retina-RPE separation results in apoptosis(Hisatomi et al. Am J Pathol. 2001 April; 158(4):1271-1278, Hisatomi etal. 2008 J Clin Invest 118: 2025-2038, herein incorporated by referencein their entireties). Retinal-detachment induced photoreceptor death wasreduced in mice carrying hypomorphic mutation of the gene encoding AIF(Hisatomi et al. 2008 J Clin Invest 118: 2025-2038, herein incorporatedby reference in its entirety).

In clinical practice, patients generally present with a detachmenthaving already occurred. The animal models of retina-RPE separation showthat Fas-pathway activation takes place early and remains elevatedthroughout the duration of the detachment (Zacks et al. Arch Ophthalmol2007; 125:1389-1395, Zacks et al. IOVS 2004; 45(12):4563-4569.8). It iscontemplated that one utility of photoreceptor-protective therapy couldbe to help prevent further photoreceptor loss until the retina could bere-attached (e.g., surgically) and normal retina-RPE homeostasisrestored. The separation of retina and RPE is also encountered in abroad spectrum of retinal diseases. It is contemplated that the clinicalrelevance of anti-Fas therapy in photoreceptor survival is not limitedto retinal detachment. For example, Fas-mediated apoptosis may play arole in photoreceptor cell death in age-related macular degeneration(AMD) (Dunaief et al. Arch Ophthalmol. 2002; 120(11):1435-1442, hereinincorporated by reference in its entirety). Age-related maculardegeneration is characterized by progressive degeneration of the RPE andcauses outer retinal degeneration and re-organization similar to thatwhich occurs after retinal detachment (Jager et al. N Engl J Med. 2008;358:2606-17, Johnson et al. Invest Ophthalmol Vis Sci. 2003;44:4481-488, herein incorporated by reference in their entireties). Inthe neovascular form of AMD there is also the exudation of fluid underthe retina, creating an actual separation of this tissue from theunderlying RPE (Jager et al. N Engl J Med. 2008; 358:2606-17, hereinincorporated by reference in its entirety). Neovascular AMD can resultin prolonged periods of retina-RPE separation and Fas-pathwayactivation. The utility of anti-Fas treatment would most likely be as anadjunct aimed at extending the survival of photoreceptors while theunderlying disorder is being treated (Brown et al. N Engl J Med. 2006Oct. 5; 355(14):1432-44, herein incorporated by reference in itsentirety). Though additional apoptotic pathways may be activated afterretina-RPE separation, experiments performed during development ofembodiments of the present invention indicate that a significant numberof photoreceptors are preserved by inhibiting the Fas death receptor. Itis contemplated that combining Met12 treatment with anotheranti-apoptotic, e.g. XIAP, or with a pro-survival molecule, e.g. IL-6,increases the efficiency of photoreceptor survival.

In some embodiments, the present invention provides compositions, kits,systems, and/or methods to prevent, inhibit, block, and/or reducephotoreceptor cell death. In some embodiments, the present inventioninhibits apoptosis of photoreceptors. In some embodiments, photoreceptordeath and/or apoptosis is caused by retinal detachment, age-relatedmacular degeneration, trauma, cancer, tumor, inflammation, uveitis,diabetes, hereditary retinal degeneration, and/or a disease affectingphotoreceptor cells. In some embodiments, the present invention enhancesphotoreceptor viability and/or inhibits photoreceptor death (e.g. duringretinal detachment and/or is ocular conditions which do not involveretinal detachment. In some embodiments, the present invention findsutility in enhances photoreceptor viability and/or inhibitsphotoreceptor death in a variety of conditions and/or diseasesincluding, but not limited to macular degeneration (e.g. dry, wet,non-exudative, or exudative/neovascular), ocular tumors, hereditaryretinal degenerations (e.g. retinitis pigmentosa, Stargardt's disease,Usher Syndrome, etc), ocular inflammatory disease (e.g. uveitis), ocularinfection (e.g. bacterial, fungal, viral), autoimmune retinitis (e.g.triggered by infection), trauma, diabetic retinopathy, choroidalneovascularization, retinal ischemia, retinal vascular occlusive disease(e.g. branch retinal vein occlusion, central retinal vein occlusion,branch retinal artery occlusion, central retinal artery occlusion,etc.), pathologic myopia, angioid streaks, macular edema (e.g. of anyetiology), central serous chorioretinopathy. In some embodiments, thepresent invention comprises administration of a composition to inhibitphotoreceptor death (e.g. apoptosis). In some embodiments, a compositioncomprises a pharmaceutical, small molecule, peptide, nucleic acid,molecular complex, etc. In some embodiments, the present inventionprovides administration of a photoreceptor protective polypeptide toinhibit photoreceptor apoptosis. In some embodiments, a polypeptide ofthe present invention can be prepared by methods known to those ofordinary skill in the art. For example, the claimed polypeptide can besynthesized using solid phase polypeptide synthesis techniques (e.g.Fmoc). Alternatively, the polypeptide can be synthesized usingrecombinant DNA technology (e.g., using bacterial or eukaryoticexpression systems). Accordingly, to facilitate such methods, thepresent invention provides genetic vectors (e.g., plasmids) comprising asequence encoding the inventive polypeptide, as well as host cellscomprising such vectors. Furthermore, the invention provides thepolypeptide produced via recombinant methods.

In some embodiments, the present invention provides administration ofphotoreceptor protective compositions (e.g. photoreceptor protectivepeptides, polypeptide, small molecules, nucleic acids, nucleic acidsencoding protective peptides, etc.). In some embodiments, the presentinvention provides administration of polypeptides which inhibitapoptosis of photoreceptor cells (e.g. IL-6, XIAP, MET, fragmentsthereof, etc.). In some embodiments, the present invention providesadministration of nucleic acids which encode polypeptides (e.g. IL-6,XIAP, MET, fragments thereof, etc.) which inhibit apoptosis ofphotoreceptor cells. In some embodiments, administered compositionsinhibit apoptotic pathways. In some embodiments, MET polypeptide isadministered (e.g. to a subject, cell or cells) as a inhibitor ofapoptosis and/or photoreceptor protective peptide. In some embodiments,MET12 polypeptide is administered. In some embodiments, a polypeptidewith at least 50% homology to MET or MET12 is administered (e.g. atleast 60% homology, at least 70% homology, at least 80% homology, atleast 90% homology, at least 95% homology, at least 99% homology, etc.).In some embodiments, a polypeptide with at least 50% homology to IL-6 isadministered (e.g. at least 60% homology, at least 70% homology, atleast 80% homology, at least 90% homology, at least 95% homology, atleast 99% homology, etc.). In some embodiments, a polypeptide with atleast 50% homology to XIAP is administered (e.g. at least 60% homology,at least 70% homology, at least 80% homology, at least 90% homology, atleast 95% homology, at least 99% homology, etc.). In some embodiments,administering a peptide, nucleic acid, or a drug-like small molecule toa subject or cell inhibits apoptotic pathways, inhibits apoptosis,and/or protects against photoreceptor death.

In some embodiments, polypeptides of the present invention are isolatedand/or purified (or substantially isolated and/or substantiallypurified). Accordingly, the invention provides polypeptide insubstantially isolated form. In some embodiments, polypeptides areisolated from other polypeptides as a result of solid phase proteinsynthesis, for example. Alternatively, polypeptides can be substantiallyisolated from other proteins after cell lysis from recombinantproduction. Standard methods of protein purification (e.g., HPLC) can beemployed to substantially purify polypeptides.

In some embodiments, the present invention provides a preparation ofpolypeptides in a number of formulations, depending on the desired use.For example, where the polypeptide is substantially isolated (or evennearly completely isolated from other proteins), it can be formulated ina suitable medium solution for storage (e.g., under refrigeratedconditions or under frozen conditions). Such preparations may containprotective agents, such as buffers, preservatives, cryprotectants (e.g.,sugars such as trehalose), etc. The form of such preparations can besolutions, gels, etc., and the inventive polypeptide can, in someembodiments, be prepared in lyophilized form. Moreover, suchpreparations can include other desired agents, such as small moleculesor even other polypeptides and proteins, if desired. Indeed, theinvention provides such a preparation comprising a mixture of differentembodiments of the inventive polypeptide (e.g., a plurality ofpolypeptide species as described herein).

In some embodiments, the present invention also provides apharmaceutical composition comprising of one or more polypeptides(including mixtures thereof) and a pharmaceutically acceptable carrier.Any carrier which can supply a polypeptide without destroying the vectorwithin the carrier is a suitable carrier, and such carriers are wellknown in the art. The composition can be formulated for parenteral,oral, or topical administration. For example, a parenteral formulationcould consist of a prompt or sustained release liquid preparation, drypowder, emulsion, suspension, or any other standard formulation. An oralformulation of the pharmaceutical composition could be, for example, aliquid solution, such as an effective amount of the compositiondissolved in diluents (e.g., water, saline, juice, etc.), suspensions inan appropriate liquid, or suitable emulsions. An oral formulation couldalso be delivered in tablet form, and could include excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. A topicalformulation could include compounds to enhance absorption or penetrationof the active ingredient through the skin or other affected areas, suchas dimethylsulfoxide and related analogs. The pharmaceutical compositioncould also be delivered topically using a transdermal device, such as apatch, which could include the composition in a suitable solvent systemwith an adhesive system, such as an acrylic emulsion, and a polyesterpatch. Compositions could be delivered via eye drops or other topicaleye delivery method. Compisitions may be delivered intraocularly,anywhere in the eye including, for example, the vitreous cavity, theanterior chamber, etc. Compisitions may be delivered intravitrealy as iscommonly done with intravitreal injections of Lucentis (ranabizumab),Avastin (bevazizumab), triamcinolone acetonide, antibiotics, etc.Compisitions may be delivered periocularly (e.g. to the tissue aroundthe eyeball (globe) but within the bony orbit). Compositions may bedelivered via intraocular implant (e.g. gancyclovir implant,fluocinolone implant, etc.). In intraocular implant delivery, devicescontaining compositions of the present invention are surgicallyimplanted (e.g. within the vitreous cavity), and the drug is releasedinto the eye (e.g. at a predetermined rate). Compositions may beadministered using encapsulated cell technology (e.g. by Neurotech) inwhich genetically modified cells are engineered to produce and secretecompositions of the present invention (e.g. Met 12). Compositions may bedelivered via transcleral drug delivery using a device sutured or placednext to the globe that would slowly elute the drug, which would thendiffuse into the eye.

In some embodiments, the invention provides a method of employingpolypeptides to attenuate the activation of one or more members of theTNFR superfamily, desirably Fas and/or TNFR in photoreceptors and/orretinas. In some embodiments, such method is employed, for example, toinhibit cell death (e.g., apoptosis) in cells and tissues, and it can beemployed in vivo, ex vivo or in vitro. Thus, the invention provides forthe use of the inventive polypeptide for attenuating cell death (e.g.retinal cell death) in accordance with such methods. For in vitroapplication, the inventive polypeptide is provided to cells, typically apopulation of cells (e.g., within a suitable preparation, such as abuffered solution) in an amount and over a time course sufficient toinhibit apoptosis within the cells or to inhibit inflammation. Ifdesired, a controlled population untreated with the inventivepolypeptide can be observed to confirm the effect of the inventivepolypeptide in reducing the inhibition of cell death or inflammationwithin a like population of cells.

In some embodiments, the methods of the present invention are employedin vivo. In some embodiments, polypeptides are delivered to a human oranimal subject in an amount and at a location sufficient to inhibit orattenuate apoptosis or inflammation within the patient (e.g., withindesired tissue). Polypeptide can be formulated into a suitablepharmaceutical composition (e.g., as described above or as otherwiseknown to those of ordinary skill in the art) for delivery into thesubject. The delivery can be local (e.g., by injection or implantationwithin the desired tissue to be treated) or systemic (e.g., byintravenous or parenteral injection).

In some embodiments, the present invention provides a method fortreating patients suffering from such retinal detachment and or retinaldisorders and in need of treatment. In some embodiments, apharmaceutical composition comprising at least one polypeptide of thepresent invention is delivered to such a patient in an amount and at alocation sufficient to treat the disorder or disease. In someembodiments, polypeptides of the present invention (or pharmaceuticalcomposition comprising such) can be delivered to the patientsystemically or locally, and it will be within the ordinary skill of themedical professional treating such patient to ascertain the mostappropriate delivery route, time course, and dosage for treatment. Itwill be appreciated that application of the inventive method of treatinga patient most preferably substantially alleviates or even eliminatessuch symptoms; however, as with many medical treatments, application ofthe inventive method is deemed successful if, during, following, orotherwise as a result of the inventive method, the symptoms of thedisease or disorder in the patient subside to a degree ascertainable.

In some embodiments, the present invention provides methods forincreasing photoreceptor survival comprising administering aphotoreceptor protective pharmaceutical composition. The pharmaceuticalcompound may be administered in the form of a composition which isformulated with a pharmaceutically acceptable carrier and optionalexcipients, adjuvants, etc. in accordance with good pharmaceuticalpractice. The photoreceptor protective pharmaceutical composition may bein the form of a solid, semi-solid or liquid dosage form: such aspowder, solution, elixir, syrup, suspension, cream, drops, paste andspray. As those skilled in the art would recognize, depending on thechosen route of administration (e.g. eye drops, injection, etc.), thecomposition form is determined. In general, it is preferred to use aunit dosage form of the inventive inhibitor in order to achieve an easyand accurate administration of the active pharmaceutical compound. Ingeneral, the therapeutically effective pharmaceutical compound ispresent in such a dosage form at a concentration level ranging fromabout 0.5% to about 99% by weight of the total composition: i.e., in anamount sufficient to provide the desired unit dose. In some embodiments,the pharmaceutical composition may be administered in single or multipledoses. The particular route of administration and the dosage regimenwill be determined by one of skill in keeping with the condition of theindividual to be treated and said individual's response to thetreatment. In some embodiments, a photoreceptor protectivepharmaceutical composition in a unit dosage form for administration to asubject, comprising a pharmaceutical compound and one or more nontoxicpharmaceutically acceptable carriers, adjuvants or vehicles. The amountof the active ingredient that may be combined with such materials toproduce a single dosage form will vary depending upon various factors,as indicated above. A variety of materials can be used as carriers,adjuvants and vehicles in the composition of the invention, as availablein the pharmaceutical art. Injectable preparations, such as oleaginoussolutions, suspensions or emulsions, may be formulated as known in theart, using suitable dispersing or wetting agents and suspending agents,as needed. The sterile injectable preparation may employ a nontoxicparenterally acceptable diluent or solvent such as sterile nonpyrogenicwater or 1,3-butanediol. Among the other acceptable vehicles andsolvents that may be employed are 5% dextrose injection, Ringer'sinjection and isotonic sodium chloride injection (as described in theUSP/NF). In addition, sterile, fixed oils may be conventionally employedas solvents or suspending media. For this purpose, any bland fixed oilmay be used, including synthetic mono-, di- or triglycerides. Fattyacids such as oleic acid can also be used in the preparation ofinjectable compositions.

In some embodiments, photoreceptor protective compositions of thepresent invention are administered optically, for example, using thetechniques described herein, and/or other techniques (e.g. injection,topical administration, etc.) known to those in the art (See, e.g.,Janoria et al. Expert Opinion on Drug Delivery. July 2007, Vol. 4, No.4, Pages 371-388; Ghate & Edelhauser. Expert Opin Drug Deliv. 2006March; 3(2):275-87; Bourges et al. Adv Drug Deliv Rev. 2006 Nov. 15;58(11):1182-202. Epub 2006 Sep. 22; Gomes Dos Santos et al. Curr PharmBiotechnol. 2005 February; 6(1):7-15; herein incorporated by referencein their entireties.

In some embodiments, photoreceptor protective compositions of thepresent invention are provided as part of a kit. In some embodiments, akit of the present invention comprises one or more photoreceptorprotective compositions and/or photoreceptor protective pharmaceuticalcompositions. In some embodiments, a kit comprises a photoreceptorprotective composition is configured for co-administration with one ormore additional compositions (e.g. pharmaceutical compositions). In someembodiments, one or more photoreceptor protective compositions areco-administered with one or more other agents for effective protectionof photoreceptors and/or inhibition of apoptosis.

EXPERIMENTAL Example 1 Compositions and Methods

Experimental Model of Retinal Detachment

All experiments performed during the development of embodiments of thepresent invention were performed in accordance with the ARVO Statementfor the Use of Animals in Ophthalmic and Vision Research and theguidelines established by the University Committee on Use and Care ofAnimals of the University of Michigan. Detachments were created in adultmale Brown-Norway rats (300-400 g) (Charles River Laboratories,Wilmington, Mass.), wildtype C57BL mice (age 3-6 weeks) (JacksonLaboratory, Bar Harbor, Me.), and IL-6−/− mice on a C57BL background(age 3-6 weeks) (Jackson Laboratory, Bar Harbor, Me.)(Zacks et al. ArchOphthalmol. 2007; 125(10):1389-1395; herein incorporated by reference inits entirety). Rodents were anesthetized with a 50:50 mix of ketamine(100 mg/mL) and xylazine (20 mg/mL), and pupils were dilated withtopical phenylephrine (2.5%) and tropicamide (1%). A 20-gaugemicrovitreoretinal blade (Walcott Scientific, Marmora, N.J.) was used tocreate a sclerotomy 2 mm posterior to the limbus, carefully avoidinglens damage. A Glaser subretinal injector (32-gauge tip; BD OphthalmicSystems, Sarasota, Fla.) was introduced through the sclerotomy into thevitreous cavity and then through a peripheral retinotomy into thesubretinal space. Sodium hyaluronate (10 mg/mL) (Pharmacia and UpjohnCo., Kalamazoo, Mich.) was slowly injected to detach the neurosensoryretina from the underlying retinal pigment epithelium. Approximatelyone-third to one-half of the neurosensory retina was detached.Detachments were created in the left eye, leaving the right eye as thecontrol. In some eyes, either 0.15 μg anti-human IL-6 neutralizingantibody (NAB) (R&D Systems, Minneapolis, Minn.) or 15 ng exogenoushuman IL-6 (R&D Systems, Minneapolis, Minn.) was injected into thesubretinal space of the detachment in a 10 μl volume at the time ofcreation of the detachment or at a subsequent time point. Doses werebased on manufacturers' recommendations based on in vitro activityassays.

Western Blot Analysis

Retinas from experimental eyes with detachments and control eyes withoutdetachments were dissected from the RPE-choroid at 3 days after retinaldetachment, homogenized, and lysed in buffer containing 10 mM HEPES (pH7.6), 0.5% IgEPal, 42 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF),1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and 5 mM MgCl₂ and 1tablet of protease inhibitors per 10 mL buffer (Complete Mini; RocheDiagnostics GmbH, Mannheim, Germany). The homogenates were incubated onice and centrifuged at 22,000 g at 4° C. for 60 minutes. The proteinconcentration of the supernatant was then determined (DC Protein Assaykit; Bio-Rad Laboratories, Hercules Calif.). The protein samples wereloaded and run on SDSPage polyacrylamide gels (Tris-HCl Ready Gels;Bio-Rad Laboratories). After electrophoretic separation, the proteinswere transferred onto polyvinylidene fluoride (PVDF) membranes(Immobilon-P; Amersham Pharmacia Biotech, Piscataway, N.J.). Proteinbands were visualized with Ponceau S staining, and the lanes assessedfor equal loading by densitometry of a nonspecific band present acrossall lanes. Membranes were then immunoblotted for phospho-STAT1 (Tyr701)or phospho-STAT3 (Tyr705) using an immunoblotting kit (PhosphoPlus®Stat1 (Tyr701) Antibody Kit #9170 or PhosphoPlus® Stat3 (Tyr705)Antibody Kit #9130, respectively, Cell Signaling Technology, Danvers,Mass.) according to the manufacturer's instructions using a 1:1000dilution of the primary antibody.

TUNEL Staining and Histology

At varying intervals after creation of the detachment, the animals wereeuthanized, and the eyes were enucleated. For TUNEL staining, whole eyeswere fixed overnight at 4° C. in phosphate-buffered saline with 4%paraformaldehyde (pH 7.4). The specimens were embedded in paraffin andsectioned at a thickness of 5-6 μm. TUNEL staining was performed on thesections with the ApopTag Fluorescein In Situ Apoptosis Detection Kitaccording to the manufacturer's instructions (Millipore, Billerica,Mass.). For light microscopic analysis, paraffin sections were stainedwith 0.5% toluidine blue in 0.1% borate buffer.

Data Analysis

Photoreceptor cell apoptosis was quantified as the percentage of totalcells in the outer nuclear layer (ONL) that was TUNEL positive. Threenon-overlapping high power fields (40×) at the maximal height of the RDwere selected per section and were averaged unless there were less thanthree non-overlapping high power fields in which case fewer fields wereused. One representative section was used per eye. The total number ofcells in the ONL was measured in a similar fashion. The total thicknessof the retina (measured from the outer edge of the ONL to the innerlimiting membrane) was measured in three places in each of threenon-overlapping high power fields (40×) at the maximal height of the RDper section and averaged for each eye. Photoreceptor inner and outersegments were not included in the total retinal thickness measurementgiven variable retraction of these elements after detachment of theneurosensory retina which does not necessarily correlate withpost-reattachment viability of the photoreceptors (Guerin et al. InvestOphthalmol Vis Sci. 1993; 34(1):175-183; Lewis et al. Invest OphthalmolVis Sci. 1995; 36(12):2404-2416; herein incorporated by reference intheir entireties). For toluidine blue stained specimens, normalizationof ONL cell count to the total retinal thickness of each section (i.e.,ONL cell count divided by total retinal thickness) was performed toaccount for possible differences in angles of sectioning and allow forinter-sample comparison. ONL cell counts and total retinal thicknessesin each group of the rat experiments were also normalized tocorresponding values of attached retinas in that group to allowinter-sample comparison. For each experimental group, measurements weredone on 3 sections from 4-11 eyes, each eye from a separate animal.

Statistical analysis comparing percentage of TUNEL positive cells in theONL between groups and comparing the ONL cell count/total retinalthickness ratio between groups was performed using 2-tailed Student's ttests without assuming equal variance.

Example 2 Interleukin-6 as a Photoreceptor Neuroprotectant in anExperimental Model of Retinal Detachment

The immediately downstream transducers of IL-6 receptor signaling areSignal Transducers and Activators of Transcription (STATs) (Heinrich etal. Biochem J. 2003; 374(Pt 1):1-20, Samardzija et al. FASEB J. 2006;20(13):2411-2413; herein incorporated by reference in their entireties).In the context of retinal-RPE separation, STAT1 and STAT3 transcript andprotein levels are increased (Zacks et al. Invest Ophthalmol Vis Sci.2006; 47(4):1691-1695, herein incorporated by reference in itsentirety). Increased phosphorylation (i.e., activation) of STAT1 andSTAT3 in detached retinas compared to attached retinas. Injection of theIL-6 neutralizing antibody into the subretinal space at the time ofdetachment resulted in approximately a 50% reduction in the level ofphosphorylated STAT3. There was not any reduction in the level ofphosphorylated STAT1. These data suggest that after retinal-RPEseparation, IL-6 effect is mediated predominantly through STAT3 but notSTAT1.

Experiments were performed during development of embodiments of thepresent invention in which retinal-RPE separation was created inwildtype C57BL and IL-6−/− mice. Three days after detachment, the eyeswere harvested, and apoptosis within the retina was evaluated with TUNELstaining TUNEL positive cells were confined to the ONL ofphotoreceptors, consistent with prior studies of experimental RD.3 Thepercentage of TUNEL positive cells in the ONL of detached retinas wassignificantly greater in the IL-6−/− mice compared to the wildtype mice(SEE FIG. 2). Detachments in wildtype and IL-6−/− mice were created andmaintained for 1 and 2 months. Eyes were then harvested for toluidineblue staining. The ONL cell count/total retinal thickness ratio was verysimilar between attached retinas of wildtype mice and attached retinasof IL-6−/− mice (SEE FIG. 3). There was a decline in the normalized ONLcell count at the 1 month time point for both wildtype and IL-6−/− miceas compared to attached retinas at time zero, but the rate ofphotoreceptor cell death was significantly higher in the IL-6−/− mice(SEE FIG. 3). Two months after detachment, there was further decline inthe ONL cell count/total retinal thickness ratio. The IL-6−/− group hadlower final ONL cell counts that wild-type animals (SEE FIG. 3).Retinal-RPE separation was created in Brown Norway rats and eithervehicle only or vehicle plus 0.15 μg anti-human IL-6 NAB was injectedsubretinally at the time of detachment. TUNEL staining of rat eyes 3days after detachment revealed significantly higher percentages of TUNELpositive cells in the ONL of retinas treated with anti-IL-6 NAB comparedto those treated with vehicle only (SEE FIG. 4 A-D, G). 15 ng ofrecombinant IL-6 was injected subretinally at the time of creation ofthe detachment. The percentage of TUNEL positive cells in the group ofrats treated with subretinal exogenous IL-6 was no different than thatof rats treated with vehicle alone, but still significantly less thanthat of rats treated with subretinal anti-IL-6 NAB (SEE FIG. 4).

When the period of retinal-RPE separation was extended to 4 and 8 weeks,subretinal injection of anti-IL-6 NAB at the time of detachment resultedin significantly lower normalized ONL cell counts 4 weeks afterdetachment compared to subretinal injection of vehicle only (SEE FIG.5B, D, H). In contrast, subretinal administration of exogenous IL-6 atthe time of detachment resulted in significantly higher ONL cellcount/total retinal thickness ratios 4 weeks after detachment comparedto controls injected with vehicle only (SEE FIG. 5B, F, H). Subretinaladministration of exogenous IL-6 appeared to slow the rate ofphotoreceptor cell loss during the first month after detachment, but therate accelerated during the second month after detachment such that theprotective benefit of exogenous IL-6 was lost, and the ONL cellcount/total retinal thickness ratios were similar between groups treatedwith vehicle only, anti-IL-6 NAB, and exogenous IL-6 8 weeks afterdetachment (SEE FIG. 5C, E, G, H).

Rats were injected with exogenous IL-6 at the time of creation of thedetachment followed by a second injection of the same dose of exogenousIL-6 at 4 weeks after detachment. At eight weeks after creating the RD,the ONL cell count/total retinal thickness was still significantlyhigher in animals with repeat IL-6 injection at 4 weeks than in controlanimals (SEE FIG. 5H).

Retinal-RPE separation was created as per the above protocol, followedby injection of exogenous IL-6 two weeks after creation of the RD. Eyeswere harvested 4, 6, and 8 weeks following detachment (i.e., 2, 4, and 6weeks following subretinal IL-6 injection, respectively) and stainedwith toluidine blue. The ONL cell count/total retinal thickness wasminimally lower in the group in which IL-6 injection was delayed 2 weeksas compared to the group in which IL-6 injection was administered at thetime of creation of the detachment, and was significantly higher thancontrol animals (SEE FIG. 5H). As with the animals treated with IL-6 atthe time of retinal-RPE separation, the effect of delayed IL-6 injectionseemed to diminish after the 4 week post-detachment time point, with thenumber of ONL cells reaching the control values by 8 weeks afterdetachment.

Example 3 Compositions and Methods Relating to X-Link Inhibitor ofApoptosis

Animals

Adult male, Brown Norway rats, at least 6 weeks of age, were purchasedfrom Harlan (Indianapolis, Ind.) and Charles River Laboratories(Wilmington, Mass.). Animals were maintained under standard laboratoryconditions and all procedures conformed to both the ARVO Statement forthe Use of Animals in Ophthalmic and Vision Research and the guidelinesof the University of Ottawa Animal Care and Veterinary Service. Ratswere divided into two groups, with one group receiving XIAP gene therapyand the other receiving the green fluorescent protein (GFP) control.

Construction of the Recombinant Adeno-Associated Virus (rAAV) Vectors

A cDNA construct encoding the full-length, open-reading frame of humanXIAP with an Nterminal hemagglutinin (HA) tag was inserted into a pTRvector under the control of the chicken β-actin promoter. A GFPconstruct was similarly generated for use as a surgical and viralcontrol. X-linking inhibitor of apoptosis (XIAP) viral transgeneexpression was enhanced by inserting a woodchuck hepatitis viruspost-transcriptional regulatory element (WPRE) in the 3′ untranslatedregion of the construct. Serotype 5 rAAV was generated (Hauswirth et al.Methods Enzymol 2000; 316:743-761, Zolotukhin et al. Methods 2002;28:158-167, herein incorporated by reference in their entireties) andpurified (Leonard et al. PLoS ONE 2007; 2:e314, herein incorporated byreference in its entirety). Viral titers were 2.33×1012 physicalparticles/ml for rAAV-GFP and 1.87×1013 physical particles/ml forrAAVXIAP. Ratios of physical to infectious particles were less than 100.

Subretinal Injections

An injection of rAAV carrying either XIAP or GFP was delivered to thesubretinal space of the left eye of each rat. The right eye served as anuntreated control. Animals were anesthetized by 2% isofluorane gasinhalation. Eyes were dilated using 1% tropicamide (Mydriacyl; AlconCanada, Mississauga, ON, Canada) and 2.5% phenylephrine hydrochloride(Mydfrin; Alcon). Proparacaine hydrochloride drops (0.5%, Alcaine,Alcon) were administered as a local anesthetic. Pain management wasachieved by buprenorphine injection (0.04 mg/kg). To maintainlubrication throughout the procedure, 0.3% hypromellose (Genteal gel;Novartis Pharmaceuticals Inc., Mississauga, ON, Canada) was applied tothe eye. Subretinal injections were performed by creating a sclerotomyapproximately 2 mm posterior to the limbus with a 20-gauge V-lance knife(Alcon). Care was taken to avoid lens contact as this could inducecataract development. A cover slip coated with Genteal was placed on topof the eye to provide magnification and visualization of the back of theeye. A 33-gauge blunt needle attached to a 10 ml syringe (HamiltonCompany, Reno, Nev.) was inserted through the scleral puncture, guidedlateral to the lens, and inserted through the retina. A 2 μL volume ofrAAV-XIAP or rAAV-GFP combined with fluorescein tracer (50:1 v/v) wasdelivered to the sub retinal space of the eye. The fluorescein allowedfor immediate visualization and evaluation of the injection location,allowing ascertainment of a successful subretinal delivery. Injectionswere delivered in a consistent manner between the 12:00 and 2:00position. Post-surgical care consisted of administration of theantibiotic 0.3% ciprofloxacin hydrochloride (Ciloxan; Alcon), and anon-steroidal anti-inflammatory drug 0.03% flurbiprofen sodium (Ocufen;Allergan, Irvine, Calif.) for five days postinjection.

Retinal Detachment

Approximately two weeks following viral injections, a retinal detachmentwas performed in the left eye of each rat. The detachments were createdby injecting 10 mg/mL sodium hyaluronate (Healon; AMO, Santa Ana,Calif.) into the subretinal space near the site of the viral injection.Approximately one-third to one-half of the retina was detached, leavingthe remaining attached portion to serve as an internal control. Animalswere sampled at 24 hours, 3 days, and 2 months after detachment.

Caspase Assays—24 Hours after Detachment

Twenty-four hours after the creation of the detachment, the intact rightretinas (internal control) and the detached portion of the left retinaswere harvested from XIAP (N=15) and GFP (N=15) animals, and protein wasextracted (Zacks et al. Invest Ophthalmol Vis Sci 2004; 45:4563-4569,herein incorporated by reference in its entirety). Caspase 9 activitywas measured using a Caspase 9 Colorimetric Assay Kit (BioVisionResearch Products, Mountain View, Calif.), as per the manufacturer'sinstructions. This assay is based on the detection of the chromophorep-nitroanilide (pNA) following cleavage from the labeled substrateLEHD-pNA. Caspase 3 activity was measured using a Caspase 3 ColorimetricAssay kit (Chemicon International, Billerica, Mass.), as permanufacturer's instructions. This assay is based on cleavage of thepNADEVD substrate by activated caspase 3.

Tissue Fixation and Processing

Rats were administered a lethal injection of Euthansol and weresubsequently perfused with 4% paraformaldehyde (PFA) in order topreserve tissue structure. Left eyes were scored with a white hot needleto enable orientation during nucleation and embedment. Eyes werepunctured with a needle to allow penetration of the fixative and wereplaced in 4% PFA overnight. The samples were then taken through a seriesof dehydration steps ending with embedment in paraffin. Eyes weresectioned at 10 mm for histological analysis.

Histological Analysis

Hematoxylin and eosin (H&E) staining was performed on 10 mm sections tolocate retinal detachments. Once a detachment was identified, subsequentslides were subjected to immunohistochemical analysis in order toconfirm the presence of XIAP or GFP. XIAP was detected using an anti-HAmouse IgG primary antibody (Roche Applied Science, Laval, QC), followedby a goat anti-mouse IgG secondary antibody (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.). GFP was detected using an anti-GFPrabbit IgG (Invitrogen, Eugene, Oreg.) followed by a goat anti-rabbitIgG (Invitrogen, Eugene, Oreg.). Rhodopsin was detected with the B630monoclonal antibody. 27 Slides were counterstained with the nuclearstain 4′,6′-diamindino-2-phenylindole dihydrochloride (DAPI). Imageswere obtained using a Zeiss Axioskop light microscope with a ZeissAxioCam HRc camera. Terminal uridine deoxynucleotidyl transferase dUTPnick end labeling (TUNEL)

Staining

TUNEL was used to compare levels of apoptosis in detached regions ofXIAP-treated versus GFP-treated samples 3 days after detachment.TUNEL-positive cells were detected using the Apoptag Peroxidase in situApoptosis Detection Kit (Chemicon, Temecula, Calif.). In order toeliminate observer bias, TUNEL-positive cells were detected using aprogram developed with the mathematical software MATLAB (version R2007a,The Mathworks Inc.). The program analyzed Photoshop images (AdobeSystems Inc.) of the outer nuclear layer (ONL) of detached retinas. ATUNEL-positive nucleus was identified, and its RGB values were recorded.The software scanned the image on a pixel-by-pixel basis to determinethe number of pixels that fell within two standard deviations of the RGBvalue s of the positive nucleus. The number of “TUNEL-positive” pixelswas then divided by the total tissue pixels to yield a ratio of“TUNELpositive” pixels for that section.

Retinal Thickness Comparison

Eyes that were sampled at 2 months after the detachment were processedfor histological analysis. Images were taken of 10 μm sections stainedwith H&E. Thickness of the ONL was measured as a ratio of the number ofnuclear layers across the ONL of the detached retina divided by thenumber of nuclear layers across the ONL of the attached portion of thesame retina. Retinal thickness varies with distance from the opticnerve, the thickness of the inner nuclear layer (INL) was used as acontrol to ensure that ONL measurements were taken at the same distancefrom the optic nerve head. For both attached and detached regions, atleast four counts were taken from each animal and averaged.

Example 4 X-Link Inhibitor of Apoptosis

Caspase Activity Assays

rAAV-GFP served as a vector and surgical control, showing rates ofphotoreceptordegeneration. There was no evidence that rAAV-GFP had aneuroprotective effect or accelerated photoreceptor degeneration.Retinal detachment in the rAAV-GFP transfected eyes (GFPOS) showed theexpected elevation in caspase 3 and 9 activity as compared to intact,nondetached retinas (SEE FIG. 6). Caspase activity levels inXIAP-treated retinas (XIAP-OS) showed no detachment-induced increase,and were comparable to their contralateral attached controls. Allcaspase activities were measured at 24 hours after the detachment, whichwas previously shown to be the time of peak caspase activity.

TUNEL Analysis

Eyes were sampled at 3 days following the creation of the retinaldetachment, and were embedded, sectioned and processed forimmunohistochemistry and TUNEL analysis. The 3 day time point was chosenbecause previous experimental studies in animal models demonstrated peakTUNEL-positive staining at 3 days after retinal detachment (Zacks et al.Invest Ophthalmol Vis Sci 2003; 44:1262-1267, Lewis G P, Charteris etal. Invest Ophthalmol Vis Sci 2002; 43:2412-2420, herein incorporated byreference in its entirety) Immunohistochemistry confirmed robustexpression from both the rAAV-GFP and rAAV-XIAP viral injections (SEEFIG. 7A, B). Antibodies for GFP or XIAP identified strong staining inthe cell bodies and inner and outer segments of the photoreceptors inthe regions of the retinal detachments. This signal did not always coverthe full detachment, and was sometimes found in attached portions of theretina, indicating that the viral transfections and the detachment didnot always completely overlap. Automated, computer-based quantificationof TUNEL staining showed that there were fewer TUNEL-positive cells inthe rAAV-XIAP eyes than in the rAAV-GFP eyes (SEE FIG. 7C-E). Althoughthe XIAP-related decrease in the number of TUNEL-positive cells did notreach statistical significance as compared to the GFP-treated eyes, theresults do correlate well with XIAP-related decrease in caspase (SEEFIG. 6).

Photoreceptor Survival in Chronic Detachment

To assess long-term structural protection of photoreceptors, histologywas conducted on eyes sampled at 2 months after the retinal detachment.Immunohistochemistry against GFP (SEE FIG. 8A, B) or XIAP (SEE FIG. 8D,E) was employed to visualize the site of rAAV virus injection and tocorrelate this with histological studies. Morphological differences wereobserved between XIAP-treated and GFP-treated retinas. The inner andouter segments of the XIAP-treated samples were generally more organizedthan those of the GFP-treated retinas, although less so than attachedregions in the same eye. Rhodopsin staining confirmed that the preservedphotoreceptors were viable and produced functional protein (SEE FIG. 8C,F). The layers of photoreceptor nuclei in the ONL were counted andcompared between the detached retinas treated with either rAAV-XIAP orrAAV-GFP and their normal attached counterparts. Counts were alwaystaken at the same distance from the optic nerve along the verticalmeridian to account for the retinal thinning that naturally occurs asone moves towards the periphery of the eye. Overall, there wassignificant preservation of the ONL in the XIAP-treated detached retinas(SEE FIGS. 8 and 9). These retinas had from 4 to 8 nuclear layers(compared to 6 to 11 in the attached regions of the same eye).GFP-treated detached retinas had from 0 to 7 nuclear layers (with 6 to14 in the attached portions of the same eyes). For each animal, a ratioof ONL nuclei in the detached relative to the attached regions wascalculated (SEE FIG. 9E).

Example 5 MET Effects on Retinal Apoptosis

In experiments performed during development of embodiments of thepresent invention, retinal detachments were created using standardizedanimal model (rat). At the time of retinal detachment, one of the threecompounds was injected (mMET, MET, DMSO). At 72 hours post-detachment,the eyes harvested and processed for TUNEL staining. MET significantlyreduced the number of TUNEL-positive cells (SEE FIG. 10) (i.e. reducedthe number of apoptotic cells).

In experiments performed during development of embodiments of thepresent invention retinal detachments were created using a standardizedanimal model (rat). At 24 hours post-detachment, the eyes harvested andprocessed for caspase 8 activity assays. Caspase 8 is the firstdownstream target of FAS receptor activation. Increased caspase 8activity means more FAS receptor activation. Alpha-Met (experimentalcompound) prevented the increase in caspase 8 activity caused by retinaldetachment (SEE FIG. 11). Similar results were observed for two morecaspases that are even farther downstream in the pathway, caspase 3 and9.

Example 6 Compositions and Methods

Retinal-RPE separation was created in Brown Norway rats by subretinalinjection of 1% hyaluronic acid. Met12, derived from the Fas-bindingextracellular domain of the oncoprotein Met, was injected into thesubretinal space at the time of separation. A mutant peptide or vehicleadministered in a similar fashion acted as inactive controls. Extrinsicapoptotic pathway was induced in 661W cells using a Fas-activatingantibody in the presence or absence of Met12. Caspase 3, caspase 8, andcaspase 9 activities were measured with calorimetric and luminescentassays in retinal extracts and cell lysates. Terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) was performed in retinalsections 3 days after separation. Histology was performed in retinalsections 2 months after retinal detachment.

Experimental Model of Retinal Detachment. Rodents were anesthetized witha 50:50 mix of ketamine (100 mg/mL) and xylazine (20 mg/mL), and pupilswere dilated with topical phenylephrine (2.5%) and tropicamide (1%). A20-gauge microvitreoretinal blade (Walcott Scientific, Marmora, N.J.)was used to create a sclerotomy 2 mm posterior to the limbus, carefullyavoiding lens damage. Under direct visualization through an operatingmicroscope, a Glaser subretinal injector (32-gauge tip; BD OphthalmicSystems, Sarasota, Fla.) was introduced through the sclerotomy into thevitreous cavity and then through a peripheral retinotomy into thesubretinal space. Sodium hyaluronate (10 mg/mL) (Pharmacia and UpjohnCo., Kalamazoo, Mich.) was slowly injected to detach the neurosensoryretina from the underlying retinal pigment epithelium. In allexperiments, approximately one-third to one-half of the supero-nasalneurosensory retina was detached. Detachments were created in the samelocation in all animals tested to allow for direct comparison of retinalcell counts. Detachments were created in the left eye, leaving the righteye as the control. In some eyes, a wild-type Met YLGA 12-mer(HHIYLGAVNYIY (SEQ ID NO:1), Met12, 50 μg), a mutant Met 12-mer(HHGSDHERNYIY (SEQ ID NO:2), mMet, 50 μg), or vehicle (DMSO) wasinjected into the subretinal space in the area of the detachment in a 10μl volume using a Hamilton syringe (Hamilton Company, Reno, Nev.)immediately after the creation of the detachment.

Cell Culture. The 661W cell line was maintained in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum, 300 mg/L glutamine, 32mg/L putrescine, 40 μL/L of β-mercaptoethanol, and 40 μg/L of bothhydrocortisone 21-hemisuccinate and progesterone. The media alsocontained penicillin (90 units/ml) and streptomycin (0.09 mg/ml). Cellswere grown at 37° C. in a humidified atmosphere of 5% CO2 and 95% air.

Caspase Activity Assays. Caspase 3, caspase 8 and caspase 9 activitieswere measured with colorimetric tetrapeptide cleavage assay kits, perthe manufacturer's instructions (Bio-Vision, Mountain View, Calif.).Total retinal protein was extracted as per a previously publishedprotocol (Zacks et al. IOVS 2003; 44(3):1262-1267, herein incorporatedby reference in its entirety). One hundred microgram of total retinalprotein from either attached or detached retinas was incubated withcaspase 3 (DEVD-pNA), caspase 8 (IETD-pNA) or caspase 9 substrates(LEHD-pNA) at 200 μM final concentration for 60 minutes. Absorbance wasmeasured at 405 nm in a microplate reader (Spectra-MAX 190, MolecularDevices, Sunnyvale, Calif.). As a negative control, retinal protein wasincubated with assay buffer without the tetrapeptide. A second negativecontrol was used in which assay buffer alone was incubated with thetetrapeptide. As a positive control, purified caspase 3, caspase 8, orcaspase 9 was incubated with the tetrapeptide alone. The caspaseactivity in the detached retina was normalized against the caspaseactivity in attached retina at the same time point. For each group, thedata represents the average caspase activity levels of several (e.g. 5)independent samples, each sample consisting of protein from 5 eyes.

In cell culture experiments, caspase 8 activity was measured by acommercially available luminescent tetrapeptide (LETD) cleavage assaykit (Promega, Madison, Wis.). The 661W cells were seeded in 96-wellplates (Nunc, Rochester, N.Y.) at 1500 cells/well for 24 hours prior totreatment. Cells were pre-treated with various concentrations of Met12,mMet, or vehicle for 1 hour prior to treatment with 500 ng/ml ofFas-agonistic Jo2 monoclonal antibody (BD Biosciences, San Jose,Calif.). Caspase 8 activity was measured at various time points byincubating the cells with the pro-luminescent substrate in 96-wellplates following manufacturer's instructions. Controls includeduntreated cells and wells with no cells. Luminescence was measured in aplate reader luminometer (Turner Biosystems, Sunnyvale Calif.).

Western Blot Analysis. Retinas from experimental eyes with detachmentsand control eyes without detachments were dissected from theRPE-choroid, homogenized, and lysed in buffer containing 10 mM HEPES (pH7.6), 0.5% IgEPal, 42 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF),1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and 5 mM MgCl₂ and 1tablet of protease inhibitors per 10 mL buffer (Complete Mini; RocheDiagnostics GmbH, Mannheim, Germany). The homogenates were incubated onice and centrifuged at 22,000 g at 4° C. for 60 minutes. The proteinconcentration of the supernatant was then determined (DC Protein Assaykit; Bio-Rad Laboratories, Hercules Calif.). The protein samples wereloaded and run on SDS-polyacrylamide gels (Tris-HCl Ready Gels; Bio-RadLaboratories). After electrophoretic separation, the proteins weretransferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P;Amersham Pharmacia Biotech, Piscataway, N.J.). Protein bands werevisualized with Ponceau S staining, and the lanes assessed for equalloading by densitometry of a nonspecific band present across all lanes.Membranes were then immunoblotted for cleaved caspase 3, cleaved caspase8, and cleaved caspase 9 (Cell Signaling Technology, Danvers, Mass.)according to the manufacturer's instructions.

TUNEL Staining and Histology. At varying intervals after creation of thedetachment, the animals were euthanized, and the eyes were enucleated.For TUNEL staining, whole eyes were fixed overnight at 4° C. inphosphate-buffered saline with 4% paraformaldehyde (pH 7.4). Thespecimens were embedded in paraffin and sectioned at a thickness of 5-6μm. TUNEL staining was performed on the sections with the ApopTagFluorescein In Situ Apoptosis Detection Kit according to themanufacturer's instructions (Millipore, Billerica, Mass.). For lightmicroscopic analysis, paraffin sections were stained with 0.5% toluidineblue in 0.1% borate buffer.

Cell Counts and Retinal Thickness Measurements. Photoreceptor cellapoptosis was quantified as the percentage of total cells in the outernuclear layer (ONL) that were TUNEL positive. Three non-overlapping highpower fields (40×) at the maximal height of the retinal detachment wereselected per section and were averaged unless there were less than threenon-overlapping high power fields, in which case fewer fields were used.One representative section was used per eye. The total number of cellsin the ONL was measured in a similar fashion. The total thickness of theretina (measured from the outer edge of the ONL to the inner limitingmembrane) was measured in three places in each of three non-overlappinghigh power fields (40×) at the maximal height of the retinal detachmentper section and averaged for each eye. Photoreceptor inner and outersegments were not included in the total retinal thickness measurementgiven variable retraction of these elements after detachment of theneurosensory retina which does not necessarily correlate withpost-reattachment viability of the photoreceptors (Zou et al. Nat. Med.2007 September; 13(9):1078-85, Guerin et al. Invest Ophthalmol Vis Sci.1993; 34(1):175-183, herein incorporated by reference in theirentireties). For toluidine blue stained specimens, normalization of ONLcell count to the total retinal thickness of each section (i.e., ONLcell count divided by total retinal thickness) was performed to accountfor possible differences in angles of sectioning and allow forinter-sample comparison. ONL cell counts and total retinal thicknessesin each group of the rat experiments were also normalized tocorresponding values of attached retinas in that group to allowinter-sample comparison. For each experimental group, measurements weredone on multiple sections (e.g., 3) from multiple eyes (e.g., 10), eacheye from a separate animal.

Example 7 In Vitro Analysis of Met12

The 661W cell line is a photoreceptor line that has been immortalized bythe expression of SV40-T antigen under control of the humaninterphotoreceptor retinol-binding protein (IRBP) promoter (Al-Ubaidi etal. J Cell Biol. 1992; 119(6):1681-1687, herein incorporated byreference in its entirety). 661W cells express cone photoreceptormarkers, including blue and green cone pigments, transducin, and conearrestin (Tan et al. Invest Ophthalmol Vis Sci. 2004; (3):764-768,herein incorporated by reference in its entirety), and can undergocaspase-mediated cell death (Kanan et al. Invest Ophthalmol Vis Sci.2007; 48(1):40-51, herein incorporated by reference in its entirety).Previous experiments conducted during development of the presentinvention demonstrate that Fas signaling plays a critical role incaspase 8 activation and photoreceptor apoptosis in vivo. 661W cellswere treated with a Fas-activating antibody (Fas-Ab). Addition of theFas-Ab resulted in cell death. Activity of caspase 8 measured in 661Wcell lysates increased with increasing concentration of Fas-Ab, peakingwith the 500 ng/ml dose (SEE FIG. 12A). 661W cells were treated with 500ng/ml Fas-Ab and measured activity levels at various time points.Caspase 8 activity was significantly increased at 48 hours in 661W cellsexposed to Fas-Ab (SEE FIG. 12B).

Met inhibits the Fas pathway. A small 12-mer peptide, Met12, containingthe amino acids surrounding the Fas-binding YLGA motif of Met protectsJurkat cells from FasL induced apoptosis (Zou et al. Nat Med. 2007September; 13(9):1078-85, herein incorporated by reference in itsentirety). 661W cells were treated with Fas-Ab in the presence of Met12or the inactive mutant peptide, mMet, in which the central 6 amino acidscontaining the YLGA motif were randomly changed. Caspase 8 activity wasdetermined 48 hours after treatment as a measure of Fas-receptor pathwayactivation. Fas-Ab induced caspase 8 activation was inhibited by Met12treatment in a dose dependent manner (SEE FIG. 12C). In contrast,treatment of cells with the mMet peptide or vehicle alone had no effecton Fas-mediated caspase 8 activation. 661W cells have an intact Fasdeath receptor pathway. Met12 peptide can inhibit Fas signaling in an invitro photoreceptor model.

Example 8 In Vivo Effect of Met12

Experiments conducted during development of the present inventiondemonstrated that retinal detachment leads to the activation of Fas/FasLpathway and caspase 8 cleavage (Zacks et al. IOVS 2003; 44(3):1262-1267,Zacks et al. Arch Ophthalmol 2007; 125:1389-1395, Zacks et al. IOVS2004; 45(12):4563-4569.8, herein incorporated by reference in theirentireties). The effect of Met12 on photoreceptor caspase 8 activation24 hours after retinal detachment was examined Rat retinas were detachedin the presence of Met12 (50 μg), mMet (50 μg), or vehicle. Caspase 8activity was significantly increased in detached retinas injected withvehicle or mMet, compared to attached retinas (SEE FIG. 13). Subretinalinjection of the Met12 peptide at the time of retinal detachment reducedcaspase 8 activity by approximately 50%. These results demonstrated thatsimilar to 661W cells, treatment of detached retinas with Met12 inhibitsFas-mediated caspase 8 activation in photoreceptors.

During rodent photoreceptor apoptosis, Fas/FasL signaling acts as anupstream regulator of the intrinsic death pathway. This is demonstratedby the reduction of caspase-9 activity after injection of neutralizingantibodies against either Fas or FasL into the subretinal space of thedetached retina (Zacks et al. IOVS 2004; 45(12):4563-4569.8, hereinincorporated by reference in its entirety). Caspase 3 and caspase 9activity levels were measured 24 hours after retinal detachment in thepresence of Met12 (50 μg), mMet (50 μg), or vehicle. Subretinal Met12injection reduced caspase 3 activity by approximately 50% after 24 hours(SEE FIG. 13). Similarly, caspase 9 activity was also reduced by about50% in detached retinas injected with Met12 (SEE FIG. 13). In contrast,subretinal injection of the mMet did not affect the activity level ofeither of these caspases. The conversion of pro-caspase 8 to cleavedcaspase 8 was confirmed on Western blot (SEE FIG. 13), as was thecleavage of caspases 3 and 9. These results demonstrated thatMet12-mediated inhibition of the Fas-receptor leads to reducedactivation of the intrinsic cell death pathway in detachedphotoreceptors.

In rodent eyes, the peak of TUNEL staining occurs at 3 days afterretina/RPE separation, with a rapid decline in TUNEL-positive cells tonear pre-separation levels by day 7 (Zacks et al. IOVS 2003;44(3):1262-1267, Zacks et al. Arch Ophthalmol 2007; 125:1389-1395,herein incorporated by reference in their entireties). Rat retinas weredetached in the presence of Met12, mMet, or vehicle. At 3 days afterretinal detachment, TUNEL positive cells were confined to the ONL ofphotoreceptors (SEE FIG. 14A). About 5% of ONL cells displayedTUNEL-positive staining at day 3 post-separation (SEE FIG. 14B).Injection of Met12 into the subretinal space resulted in ˜77% fewerTUNEL-positive photoreceptors as compared with separated retinasinjected with mMet (SEE FIG. 14B). No gross histological change could bedetected due to injection of the vehicle alone.

Rat retinas were detached as described above and Met12, mMet, or vehiclewas injected in the subretinal space at the time of detachment. After 2months, detached retinas showed significant reduction in ONL thicknessas compared with attached retinas (SEE FIG. 15A). Rat retinas injectedwith Met12 showed 37% increase in ONL cell counts (SEE FIG. 15B) and 27%increase in ONL thickness measurements (SEE FIG. 15C) compared withmMet-injected retinas after 2 months of retinal detachment. Theseresults demonstrated that inhibition of Fas signaling and caspaseactivation by Met12 increases the survival of photoreceptors afterprolonged retina/RPE separation.

I claim:
 1. A method of inhibiting photoreceptor apoptosis comprisingadministering to a subject a photoreceptor protective compositioncomprising a photoreceptor protective polypeptide comprising: a) afragment of MET comprising SEQ ID NO:1(MET12), or (b) a fragment of METthat comprises at least 70% sequence similarity to MET12, wherein saidpolypeptide inhibits FAS-mediated photoreceptor apoptosis, and whereinsaid photoreceptor protective composition is administered to saidsubject intraocularly, intravitrealy, or periocularly.
 2. A method ofinhibiting photoreceptor apoptosis comprising administering to a subjecta photoreceptor protective composition comprising a photoreceptorprotective polypeptide comprising: a) a fragment of MET comprising SEQID NO:1(MET12), or (b) a fragment of MET that comprises at least 70%sequence similarity to MET12, wherein said polypeptide inhibitsFAS-mediated photoreceptor apoptosis, and wherein said subject suffersfrom an ocular condition, disease, or condition or disease affectingocular health.
 3. The method of claim 2, wherein said subject suffersfrom abnormal retina-RPE (retinal pigment epithelium) homeostasis. 4.The method of claim 3, wherein said photoreceptor protective compositioninhibits photoreceptor apoptosis until normal retina-RPE homeostasis isrestored.
 5. The method of claim 2, wherein said ocular condition,disease, or condition or disease affecting ocular health comprisesretinal detachment, macular degeneration, retinitis pigmentosa, ocularinflammation, autoimmune retinopathy, trauma, cancer, tumor, uveitis,hereditary retinal degeneration, diabetic retinopathy, choroidalneovascularization, retinal ischemia, pathologic myopia, angioidstreaks, macular edema, or central serous chorioretinopathy.
 6. Themethod of claim 5, wherein said ocular condition, disease, or conditionor disease affecting ocular health comprises macular degeneration. 7.The method of claim 5, wherein said photoreceptor protective compositionis administered in an amount sufficient to enhance photoreceptorsurvival within said subject.